This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Electrostatic interactions influence the structure, function, and interaction of proteins with other biomolecules. Thus, methods that probe electrostatics provide complementary information to methods sensitive to dynamics and will aid in the construction of models for interpreting spectra. The availability of high-field/high-frequency-ESR (HFHF-ESR) allows the use of nitroxide spin labels as an accurate probe of local electrostatics in biomolecules. When the electronic energy levels are shifted by interactions with a local electric field, second-order shifts appear in the nitroxide's g-tensor. The excellent g-factor discrimination at high field permits the measurement of these small shifts that are not detectable at standard ESR fields. The sensitivity to local electrostatic effects may be greatly enhanced by the application of a strong external electric field. The possibility of exploiting the high g-factor resolution of HFHF-ESR to observe such effects in organic radicals greatly extends the range of systems that can be examined. The probe necessary for applying a strong local electric field in a high-frequency sample resonator is a natural extension of techniques we have developed for studying electric-field effects in liquid crystals. We are optimizing the thin-film electrode geometry for our standard Fabry-P[unreadable]rot (FP) resonator, to permit studies in the E parallel configuration. Development work on a series of shunt FP resonators for use at 95, 170 and 240GHz with variable coupling is underway utilizing time-domain analysis methods developed in the center. These new resonators will allow us to perform experiments in the E perpendicular configuration. The experience we have gained with the center's 95GHz pulse spectrometer has given us important insights into the sample holder requirements that will be necessary for the success of the LEFE experiments.